Fluorescent dyes

ABSTRACT

The present invention describes a class of dyes for use in staining cell samples and methods of making such dyes. A preferred class of dyes known as detergent dyes which possess the ability to stain cells in whole blood and are only slowly leached or lost from the stained cells over time are described. The present invention has application, for example, to blood typing for the determination of the presence of blood group antigens A, B, AB, O, and D (Rh o ) and antibodies to such antigens.

This is a continuation-in-part of copending application U.S. Ser. No.645,458 filed Aug. 28, 1984, now U.S. Pat. No. 4,748,129, incorporatedherein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

There is a continuing need for improved assay methods and reagents forthe detection of an analyte in a sample. The analyte can be a member ofa specific binding pair ("sbp") consisting of ligand and its homologousreceptor. Exemplary of sbp members are antigens and antibodies.

In general, a sbp member complementary to the antigen will be used as anassay reagent. Other reagents used in detection assays must haveparticular characteristics in order to be suitable for use in the assay.When the analyte is on the surface of a particle or can be caused tobind to a particle, it is desirable to use a dye, for example afluorescent dye, capable of staining the particle in a reproduciblemanner without interfering with the reaction between the analyte and itshomologous sbp member.

Mammalian red blood cells carry numerous antigens some of which must beaccurately identified in both patient and donor for medical proceduressuch as transfusions. Accurate determination of blood groups, A, B, AB,O and D (Rh_(o)) is critically important. Also, antibodies to such bloodgroup antigens as well as other circulating antigens can be ofdiagnostic interest.

Conventionally, agglutination techniques are performed on a microscopeslide or in a tube with the results being assessed visually. Improvedrapid, accurate and automated screening of blood is desirable in view ofthe critical nature of the assays performed, the labor intensity of suchassays and the large numbers of samples which must be tested.

BACKGROUND OF THE INVENTION

Researchers in the field have used such terms as squarate, squaraine andsquarylium to describe the various compounds which have as their rootsquaric acid. ##STR1##

For purposes of this invention, reference to the works of others willuse the terminology as used in the particular reference. In describingthe instant invention, the term "squaraine" will be used when referringto the squaraine compound which is derived from squaric acid andmodified so as to possess the desired fluorescent dye properties.

Various squaric acid dyes are discussed by Sprenger, et al., Angew.Chem., 80, 541 (1968); Sprenger, et al., Angew. Chem., 79; 581, 1967;Sprenger, et al., Angew. Chem. internat. Edit., 5:894 (1966); and Maaks,et al., ibid., 5:888 (1966).

Novel squaraine dyes having absorption maximums of greater than 600 nmand possessing either hydrophilic or lipophilic properties are describedin the pending U.S. application, Ser. No. 834,168 filed Feb. 27, 1986.

Assays for ligands and receptors employing the novel squaraine dyes aredescribed in pending U.S. application, Ser. No. 773,401 filed Sept. 6,1985. Use of fluorescent beads conjugated to a receptor for thescreening of red blood cells is described in U.S. Pat. No. 4,550,017issued Oct. 29, 1985.

Identification of red blood cell antigens by agglutination techniques isstandard, e.g., C. Hudson and F. C. Hay, Practical Immunology, SecondEdition, Blackwell Scientific Publications, Oxford (1980). U.S. Pat. No.3,862,303 is exemplary of immunological detection and identification ofserological factors using carrier particles such as latex beads. Smith,FEBS Letters 77,25 (1977) describes a fluorescent immunoassay.

More recently squaric acid compounds for use in the preparation ofelectrophotographic plates of use in electrophotographic imaging systemshave been described. In particular, squaric acid dyes havingphotoconducting abilities are described in U.S. Pat. No. 4,500,621issued Feb. 19, 1985. Use of more squaraine compositions inphotoresponsive devices having sensitivity to infrared and visibleillumination is described in U.S. Pat. No. 4,508,803 issued Apr. 2,1985. In particular, fluorobenzylamino derivatives of squaric acid aredescribed.

In yet another use of squaric acid compounds in electrophotography,squarylium compounds having straight chain alkyl groups with a hydroxylor halogen atom attached are described in U.S. Pat. No. 4,707,427 issuedNov. 11, 1987.

SUMMARY OF THE INVENTION

Fluorescent agents are provided for determining the presence in a sampleof a member of a specific binding pair ("sbp member") consisting ofligand and its homologous receptor. The fluorescent agents are squarainedyes which can readily be absorbed by cells in whole blood or insuspension in other aqueous media. In one embodiment of the invention,the squaraine dye is from the class of squaric acid dyes referred toherein as detergent dyes. The dye is so designed as to enter the cell inthe presence of proteins and other plasma components and to be retainedby the cell. Leaching out of the dye occurs slowly if at all.

The squaraine dye of the invention is a compound of the formula:##STR2## wherein:

R₁ and R₂ are independently selected from the group consisting of loweralkyl (1-5 carbon atoms) with the proviso that at least one of the R₁and R₂ groups has a substituent selected from the group consisting of--SO₃ H, --OSO₃ H, --PO₃ H₂, --OPO₃ H₂, --COOH and --NHSO₃ H;

R₃ and R₄ are independently selected from the group consisting of alkyl(5-15 carbon atoms); and

R₅ and R₆ are independently selected from the group consisting ofhydrogen, hydroxyl, and lower alkoxyl (1-5 carbon atoms).

In the method, the sample believed to contain an sbp member is combinedin an aqueous medium with a complementary sbp member wherein at leastthe sbp member or the complementary sbp member is bound to the surfaceof a cell. The cell is stained with a fluorescent agent, i.e. thesquarine dye, capable of being incorporated into the cell. The presenceof the sbp member is indicated by a change in fluorescence of the cellsuspension as a result of agglutination of the cells.

The present invention has particular application to blood typing, forexample, for the determination of the presence of blood group antigensA, B, AB, O, and D (Rh_(o)) and antibodies to such antigens, as well asantibodies to antigens M, N, S, s, Lewis, Lutheran, Kell, Duffy, Kidd,etc.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The subject invention provides novel reagents and methods of making suchreagents for determining the presence of an analyte, usually a sbpmember, in a sample. The method of detection employs a complementary sbpmember where at least one of either the sbp member or the complementarysbp member is bound to the surface of a cell. Also employed in themethod is a fluorescent cell incorporative agent.

Preferably, when the sbp member in the sample is not bound to thesurface of a cell and the sample contains cells, the fluorescent agentis combined with the sample by first incorporating the agent into cellsbound to a complementary sbp member and then combining the combinationwith the sample. When the sample does not contain cells, the dye can beadded before or after combining the sample with cells, or by combiningthe sample with stained cells. When the sbp member in the sample isbound to the surface of a cell, the fluorescent agent can be added tothe sample either prior to or after combining the sample with thecomplementary sbp member. Preferably, the fluorescent agent is added tothe sample prior to combining the sample with the complementary sbpmember. Thus, the term "combining with the sample" is meant to includecombining together two or more of the reagents mentioned above prior tocombining any remaining reagents. The term "reagents" includes thesample, the complementary sbp member (bound or not bound to the cells),and the fluorescent cell incorporative agent, and may further includeany additional agents required for the successful operation of thesubject method.

Before proceeding further a number of terms will be defined.

"Fluorescent cell incorporative agent"--a compound of molecular weightless than 2000 capable of being incorporated into a cell and therebycausing the cell to be fluorescent, for example, a cell membrane solubledye, a DNA intercalating dye, a vital dye or the like.

"Sbp member"--a member of a specific binding pair consisting of twodifferent molecules, where one of the molecules has an area on thesurface or in a cavity which specifically binds to a particular spatialand polar organization of the other molecule. The two members of aspecific binding pair are referred to as ligand and receptor(antiligand) and are also referred to as complementary or homologous.

"Ligand"--any organic compound for which a receptor naturally exists orcan be prepared;

"Receptor" (antiligand)--any macromolecular compound or compositioncapable of recognizing (having an enhanced binding affinity to) aparticular spatial and polar organization of a molecule, i.e., epitopicor determinant site. Illustrative receptors include naturally occurringreceptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fabfragments, lectins, and the like. The term antibody is employed in thiscase as illustrative of, and to more generally denote, receptor.

"Analyte"--the compound or composition to be measured, which is a sbpmember and may be a ligand, which is mono- or polyvalent, that is,having one or a plurality of determinant sites, haptenic and antigenic,a single compound or plurality of compounds which share at least onecommon epitopic or determinant site; or a receptor.

"Complementary sbp member"--the homologous member of a specific bindingpair where the sbp member is an analyte.

"Cell"--any one of the minute protoplasmic masses which make uporganized tissue, comprising a mass of protoplasm surrounded by amembrane including nucleated and unnucleated cells and organelles.

The fluorescent cell incorporative agent is preferably more fluorescentafter incorporation into a cell. It may be capable of incorporation intothe cell by virtue of being soluble in the cell membrane or of beingtransportable across the cell membrane and undergoing a chemicalreaction that inhibits transport out of the cell. Fluorescent agentswith high protein or carbohydrate affinity may also be useful in thepresent invention. Where the cells possess deoxyribonucleic acid (DNA),one may also employ fluorescent agents having an affinity for DNA. Thefluorescent cell incorporative agent may be a hydrophobic dye that canbe rendered water soluble by binding to a hydrophilic carrier.

The fluorescent cell incorporative agent should preferably have anabsorption maximum greater than 450 nm, more preferably greater than 540nm, to provide maximum avoidance of biological interference. For themost part, the absorption wavelength maximum should be 320 to 1000 nm,preferably 600 to 800 nm.

The molar extinction coefficient for the fluorescent cell incorporativeagent at the wavelength of the exciting light should be as high aspractical and should be greater than 1,000, preferably greater than10,000, most preferably greater than 100,000 liter mole⁻¹ centimeter⁻¹.Fluorescent cell incorporative agents are chosen to have a high quantumyield, normally greater than 0.05, preferably greater than 0.3 whenincorporated in cells. The excitation wavelength is chosen to minimizebackground fluorescence from the sample, maximize fluorescence of thestained cells, and maximize the intensity of the light source andreliability of the filters. Particularly advantageous wavelengths are490 nm and 525 nm, and 633 nm because of the availability of these wavelengths from Argon, and Helium/Neon (He/Ne) lasers, respectively. Ingeneral, longer wavelengths minimize background. A He/Ne laser tuned to633 nm is particularly desirable and dyes with a high quantum yield anda high molar coefficient of extinction at 633 nm are thereforepreferred.

In addition, it is desirable that the fluorescent cell incorporativeagent have an emission maximum at a wavelength that is preferably atleast 15 nm, more preferably at least 30 nm longer than the excitationwavelength to be used. In general, it is preferred that there be asubstantial spread or difference in wavelengths for such fluorescentagent between its absorption maximum and emission maximum.

The fluorescent agent should remain substantially incorporated in thecell during the time of the assay, particularly where cells containingsuch fluorescent agent are to be mixed with a sample containing othercells. Furthermore, it is preferable that the fluorescent cellincorporative agent not be substantially quenched when incorporated intoa cell relative to when it is in an aqueous environment. That is, theproduct of the extinction coefficient and the quantum yield at a givenexcitation wavelength should not be greatly reduced when the fluorescentagent is incorporated in the cell relative to when the fluorescent agentis not incorporated in the cell. Although an increase in fluorescence isnot required, it is usually preferable that the fluorescence of the cellincorporated agent when in the cell be at least one third, preferably atleast equal to that of the unincorporated fluorescent agent.

A further characteristic of the fluorescent cell incorporative agent isthat it not interfere with binding of the sbp members, e.g., binding ofthe antigen and antibody. The fluorescent cell incorporative agentshould preferably also exhibit a high affinity for the cell.

The number of fluorescent cell incorporative agent molecules per cellshould be sufficient to conduct a meaningful assay, generally beingabout 10² to 10⁷ of such molecules per cell, preferably 10³ to 10⁶ ofsuch molecules per cell.

As mentioned above, a preferred class of fluorescent cell incorporativeagents comprises fluorescent dyes that are soluble in the cell membrane,which means that the dyes are hydrophobic and will usually beamphiphilic to provide for sufficient water solubility to permit theagent to be incorporated in the cells in a reasonable time when added toan aqueous suspension of the cells. A preferred group of membranesoluble dyes includes certain squaraine dyes that have an absorptionmaximum greater than 600 nm and an appropriate molar extinctioncoefficient and Stokes' shift.

A preferred class of fluorescent cell incorporative agents comprise dyesthat will stain cells in substantially undiluted whole blood; that is,blood that is less than two fold diluted. A particularly preferredsubclass of these dyes are not released from the cells to a significantextent during the course of an assay in which the cells are diluted orresuspended in a different medium. Thus, on incubation with neat plasmathe fluorescence of the stained cells will decrease by less than 30% in10 minutes, preferably less than 20% in 20 minutes, most preferably,less than 10% in 30 minutes.

Exemplary of such dyes by way of illustration and not limitation arethose of the formula: ##STR3## wherein

R₁ and R₂ may be unsubstituted and independently selected from the groupconsisting of lower alkyl (1-5 carbon atoms) or substituted with one ormore substituents selected from the group --SO₃ H, --COOH, --PO₃ H₂,--OSO₃ H, OPO₃ H₂ and salts thereof; and

R₃ and R₄ are each independently selected from the group consisting ofalkyl of from 5 to 15 carbon atoms; and

R₅ and R₆ are independently selected from the group consisting ofhydrogen, hydroxyl and lower alkoxyl (1-5 carbon atoms).

Squaric acid dyes wherein the substituents on the R-groups are loweralkyl groups are disclosed by Sprenger, et al., Angew. Chem. Internat.Edit., 5:894, 1966.

A class of squaric acid dyes of the general formula described abovewhere R₁ is substituted with one or more substituents selected from thegroup --SO₃ H, --COOH, --PO₃ H₂, --OSO₃ H, OPO₃ H₂ or salts thereof arecalled "detergent dyes". This class of squaraine compounds is preferablyprepared by coupling of squaric acid to two groups of very differenthydrophilicity. These detergent dyes contain groups that can ionize toform a cation or anion, preferably an anion, most preferably a sulfonicacid, and have absorbance maxima above 600 nm with high extinctioncoefficients and high fluorescence quantum yields.

The detergent dyes are conveniently synthesized in the presence of apolar aprotic solvent and a conventional solvent. Conventional solventsinclude lower alcohol:aromatic solvent mixtures, for example,n-butanol:benzene, n-butanol:toluene and the like. Preferably, includedwith the alcohol:aromatic solvent mixture are diols, such as ethyleneglycol or propylene glycol or suitable polar aprotic solvent, forexample, dimethylsulfoxide (DMSO), dimethyl formamide (DMF),dimethylacetamide, dimethyl sulfone, sulfolane, and hexamethylphosphorictriamide. More preferably, DMSO or ethylene glycol is used as acosolvent in the reaction mixture. Use of DMSO results in the efficientcoupling of squaric acid with two groups possessing very differenthydrophilicity. In one of the preferred synthesis reactions, DMSO isused to produce a squaraine dye of the formula given above wherein R₃and R₄ are hydrocarbon chains and R₁ and/or R₂ are further substitutedwith a sulfonic acid. Detergent dyes having unsubstituted hydrocarbonchains of from 5 to 8 carbons are preferred.

While the detergent dyes can be prepared using standard conditions,i.e., refluxing n-butanol-benzene or n-butanol-toluene, the addition ofa polar aprotic solvent, such as DMSO, in the synthesis has been shownto greatly enhance the efficiency of coupling with the squaric acid.Addition of from 5-30% (vol.:vol.) of DMSO, preferably 10-20%, has beenshown to enhance the synthesis of the detergent dye. Illustrativedescriptions of the synthesis and purification of the detergent dyes areset forth in Examples 2, 3 and 4.

Desirably, a dye for use in the assay of this invention must fulfill thefollowing requirements: an absorbance perferably greater than 600 nm; anextinction coefficient of at least 150,000 liter mole⁻¹ centimeter⁻¹ ; afluorescent quantum yield of at least 0.1; the ability to stain cells toprovide a source of fluorescence; no interference with the antigens ofinterest; and, the ability to remain in the cell during the assay. As aclass, the detergent dyes meet these requirements exceptionally well.

In carrying out one embodiment of the present method, the sample, a sbpmember complementary to the analyte, and the fluorescent cellincorporative agent are combined in an aqueous assay medium and a changein fluorescence of the mixture as a result of agglutination of the cellsis then determined. The presence of the analyte in the sample isindicated by this change in fluorescence.

The present method is adaptable to a wide variety of assaydeterminations for a wide variety of sbp member analytes. It is ofspecial interest where at least one of the analyte and its complementarysbp member is a normal component of the cell surface. Cell surface sbpmembers include naturally occurring membrane components such asantigens, cell wall antigens, particularly bacterial cell walls, cellsurface receptors including receptors for activating, growth, andinhibition factors, antibodies, HLA antigens, Fc receptors, hormonereceptors, ion channels, glycolipids, lipoproteins, complementcomponents, viral antigens, membrane bound enzymes, peptidoglycan,fungal antigens, idiotypic antigens and the like. Cell types of interestinclude leukocytes, bacteria, fungal cells, erythrocytes, gametocytes,reticulocytes, lymphocytes including monocytes, macrophage, B cells, Tcells, eosinophils, etc. A particular adaptation of the present methodis in the area of blood typing. Blood group antigens, as well asantibodies thereto, may be detected using the method described above.

The subject invention provides a novel and particularly useful methodfor typing red blood cells or identifying red blood cell antigens andthe antibodies thereto by using the red blood cells as a carrier ofincorporated fluorescence where the cells agglutinate during the assaymethod. A change in fluorescence as a result of the agglutination isdetermined and is an indication of the presence of a particular redblood cell antigen or antibody thereto. Substances which bind to redblood cell antigens, normally antibodies or lectins, are required tocause agglutination of the cells. In one embodiment of the presentinvention for determining blood group antigens, whole blood is combinedwith a fluorescent cell incorporative agent, usually a squarainecompound of the above formula where R₃ and R₄ are unsubstituted, andpreferably R₁ -R₄ are ethyl, and a receptor for the antigen of interestin an aqueous medium, e.g., an appropriate buffer. If the antigen ofinterest is present on the surface of the red blood cells agglutinationof the cells will occur and a change in fluorescence will be observed asan indication of the presence of the antigen of interest.

The receptor which is employed binds preferentially to the blood groupsurface antigens of interest. Thus, there will be a fluorometricallymeasurable change when a given antigen is present as compared to whenthat antigen is absent in a given red blood cell sample. For example, inthe A, B, O system, if anti-A antibody were used, agglutination wouldoccur and there would be a change in fluorescence if the analytecontained the A antigen of type A or type AB blood over that where theanalyte contained blood types B or O.

In addition to antibodies, certain lectins are known to bindspecifically to red blood cell surface antigens, and are convenientreceptors for use in the present assays.

The compounds and methods described herein can also be used fordetermining the presence of antibodies to a red blood cell antigen. Inthis approach, red blood cells having the surface antigen homologous tothe antibody in question are employed in the assay. The antigen-bearingcells and a fluorescent agent are preferably combined first to providestained cells which are then combined with the sample in an aqueousmedium. Staining will frequently be achieved with a detergent dye of theinvention, preferably where R₃ and R₄ are octyl or heptyl, R₁ is ethyland R₂ is trimethylene sulfonic acid. Alternatively, a squaraine dye ofthe invention, where R₁ -R₄ are independently selected from the groupbutyl, pentyl and hexyl can be used for antibody screening assays. Ifthe antibody in question is present in the sample, a change influorescence will occur as the result of agglutination.

The present method is of particular importance in cross-matchdeterminations. In such a determination, blood cells from a potentialdonor and plasma from a recipient are mixed in accordance with themethod of the invention. Preferably, whole blood from the donor willfirst be combined with the fluorescent agent in order to stain the cellsand the mixture will then be combined with recipient plasma.Alternatively, the fluorescent agent may be added after combining theblood and plasma. After incubation, the cells are separated, washed, andsuspended and incubated in a solution that contains anti-immunoglobulin.A positive signal caused by agglutination indicates that the match isincompatible. The successful use of this method requires the fluorescentagent be a detergent dye. Most preferably, the detergent dye is of thegeneral formula described above where R₃ and R₄ are octyl or heptyl, R₁is ethyl and R₂ is trimethylene sulfonic acid.

The method described herein is simple and can be performed in areasonably short period of time. A particular advantage of the presentmethod is that cells in whole blood can be stained directly with noprior separation or washing of donor cells. Cells stained by this methodretain the fluorescent dye during the subsequent washing, suspending,and separation steps. The assay medium or mixture may, therefore, beobserved directly for a change in fluorescence as a result ofagglutination of the cells.

A method utilizing the dyes of the invention will next be described indetail using a blood sample as exemplary of the assay sample and bloodgroup antigens, or antibodies thereto, as exemplary of sbp members thatmay be determined in accordance with the present method. Thisdescription is by way of illustration only and is not meant to limit thescope of the present invention.

In one procedure for carrying out an assay for a blood group antigenwhich is a sbp member in accordance with the present invention, a bloodsample optionally in a buffered aqueous medium comprising greater than5%, preferably greater than 20%, more preferably greater than 50%, bloodby volume is employed. The pH of the buffered aqueous medium is usuallyabout 5 to 9, preferably about 6 to 8. The sample is mixed withappropriate amounts of a fluorescent cell incorporative agent and a sbpmember complementary to the blood group antigen, which amounts generallyshould be sufficient to result in a meaningful assay. The amount of thefluorescent cell incorporative agent depends upon the nature of thecells and the nature of the agent. Usually, about 0.1 to 100 μg,preferably about 1 to 10 μg, of fluorescent cell incorporative agent areemployed per ml of blood. The amount of complementary sbp memberemployed is determined empirically and is usually between 0.01 and 1000or more times the amount of blood group antigen, preferably 0.1 to 100times the amount of blood group antigen.

Where the fluorescent cell incorporative agent has low water solubility,a solution of the fluorescent agent in a suitable organic polar solventwill be added to the blood sample. Normally the volume of the addedsolution will be 5% less of that amount of the blood sample, preferably3% or less. The organic solvent will generally have from 1 to 6 carbonatoms and from 1 to 5 heteroatoms selected from the group consisting ofoxygen, nitrogen, and sulfur. Exemplary of such solvents aredimethylformamide, dimethylsulfoxide, hexamethylphosphoramide, and thelike. The mixture is then combined with the complementary sbp member.

The sample, fluorescent cell incorporative agent, and complementary sbpmember are combined and incubated under conditions, usually mixing, thatwill provide for agglutination of the cells when the blood group antigenof interest is present. Incubation times may vary widely depending onthe density of the blood group antigen on the cell surface, theconcentration of the cells and the complementary sbp member and thereaction conditions including the addition of agglutination enhancerssuch as polybrene, dextran or dextran derivatives, low ionic strengthmedium, serum albumin, polyethyleneglycol, and the like. Desirableincubation times are about 10 to 600 sec, preferably about 10 to 200sec, at mild temperatures usually about 10° to 37° C.

In reverse blood grouping for the determination of the presence ofantibodies to a particular blood group antigen in a sample, a sample ofplasma or whole blood is combined in an aqueous buffered medium with redblood cells of the particular type, A or B, of interest. The fluorescentcell incorporative agent is incorporated into such cells prior tocombining with the sample when the sample is whole blood, but can beadded after combining the sample with the cells when the sample isplasma. Incorporation of such agent into the red blood sample is carriedout as described above. The medium is then held for a period and underconditions for agglutination as mentioned above.

Following the above holding period, the medium is examined to determineany change in fluorescence as a result of agglutination of the cells.

Changes in fluorescence may be measured in various ways. In oneprocedure, agglutination produces a decrease in fluorescence because acellular component such as hemoglobin absorbs more incident or emittedlight from a cell in a cell cluster than from a cell that isunassociated with other cells. Other methods depend upon thedistribution of fluorescent molecules as a result of aggregation.

To this end one may use a non-flow cytometric technique known in theart, in which a small diameter beam of light produced by means of slitsor preferably a laser is used to detect high local concentration offluorescent molecules. This technique employs fluorescent pulse heightanalysis or correlation of fluorescence fluctuations: Briggs, et al.,"Homogeneous Fluorescent Immunoassay," Science, 212:1266 (1981) andNicoli, et al., "Fluorescence Immunoassay Based on Long TimeCorrelations of Number Fluctuations," Proc. Natl. Acad. Sci. USA,77(8):4904 (1980).

A preferred method for determining a change of fluorescence inaccordance with the present invention involves the use of the fiberoptic cytometer described in U.S. Pat. No. 4,564,598 issued Jan. 14,1986 and having the same assignee as the present invention, thedisclosure of which is incorporated herein in its entirety. In U.S. Pat.No. 4,564,598, method and apparatus are provided for determining thepresence of particles in a dispersion in relation to the detection ofthe presence or amount of the material of interest. An optical fiber isused to define a relatively small volume from which fluorescent lightcan be received and counted. The volume is related to the volume inwhich there is likely to be one or relatively few particles whichproduce predetermined fluctuations. By employing any one of severalmathematical techniques for fluctuation analysis, the fluorescencefluctuations are related to the presence of an analyte in a sample. Thefluctuations are observed over a period of time in a static mode orpreferably by sampling a plurality of volumes in the sample. Bycomparing the observed results with results obtained with samples orcalibrators known to be free of analyte and other samples or calibratorsknown to contain the analyte, the presence or absence of analyte inunknown samples can be determined.

As a calibrator blood grouping, a known amount of antibody or cellsbearing the appropriate blood group antigen in question is incorporatedinto an appropriate medium and treated as described above for the samplecontaining the unknown analyte. The change in fluorescence for thecalibrator is compared with the change in fluorescence for the unknownsample as an indicator of the presence of the blood group antigen orantibody thereto in question.

The fluorescent dyes of the invention can be provided in a kit inpackaged combination with predetermined amounts of reagents for use inthe detection of serum antigens. A representative kit would include thefluorescent dye and an sbp member complementary to the analyte.Alternatively, the kit could contain cells having a complimentary sbp ontheir surface which are stained with the fluorescent dye. In addition,other reagents necessary to detect the sbp member of interest in thesample and additives such as ancillary reagents may be included. Therelative amounts of the various reagents may vary widely, to provide forconcentrations in solution of the reagents which substantially optimizethe sensitivity of the assay. The reagents will usually be provided asliquid suspensions of particles but when no cells are included they canbe provided as dry powders, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing the assay.

EXAMPLES

The examples which follow are illustrative and are not limiting of theinvention.

EXAMPLE 1 Preparation of2-(p-diethylamino-m-hydroxyphenyl)-4-(diethylimmonio-2-hydroxyl-2,5-cyclohexadienylidene)-3-oxo-1-cyclobutenolate(DEAS)

DEAS was prepared as follows: squaric acid (741 mg, 65 mmole) was mixedwith stirring with 2.16 g, 13 mmole 3-N,N-diethylaminophenol in 90 ml ofn-butanol:toluene (2:1). The mixture was refluxed overnight withazeotropic removal of water. Progress of the reaction was followed bythin layer chromatography (TLC) using methanol:toluene (1:9). Next, thereaction mixture was distilled to remove about 40 ml toluene, followedby cooling of the reaction mixture to room temperature. Crystallineproduct was separated and dried at room temperature to give 2.5 g ofproduct. UV (DMF) λmax 650 nm, ε=240,000, fluorescence (DMF).

EXAMPLE 2 Preparation of1-[4-[N-ethyl-N-(3-sulfopropyl)amino]-2-hydroxyphenyl]-3-[4-diheptylamino-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediyliumdihydroxide, bis(inner salt) (C₇ SAS) ##STR4## Preparation of StartingMaterials

3-[N-ethyl-N-(3-hydroxyphenyl)amino]propane sulfonic acid was preparedas follows:

Propane sultone (1.22 g,10 mmol) and 3-(ethylamino)phenol (1.37 g, 10mmol) were warmed together in a 25 ml round bottom flask until meltingbegan. After 5-10 minutes, 2 ml of methanol was added the liquifiedmixture. After 30 minutes, additional methanol was added (5-10 ml) andthe mixture was heated to reflux. The dark lower layer solidified into apale pink powder.

The solid was washed on a glass frit with hot methanol. It was thentaken up in methanol at reflux and allowed to cool and crystallize.Dissolution in boiling methanol occured slowly with the slow formationof crystals. The resultant solid (2.04 g, 78%) was a single spot on TLC(silica, CH₃ CN/H₂ O 88:12 v/v; UV detection: R_(f) 0.59).

m-(N,N-diheptylamino)phenol hydrochloride was prepared as follows:

A mixture of 3-aminophenol (3 g, 0.034 mole, Aldrich Chemical Co.,Inc.), 1-iodoheptane (15.6 g, 0.069 mole, Aldrich Chemical Co. Inc.) andN,N-diisopropylethylamine (13.4 g, 0.104 mole, Aldrich Chemical Co.,Inc.) in methanol (17 ml) was refluxed under nitrogen for 28 hours. Thereaction product (R_(f) 0.55, silica, ethylacetate:hexane 1:4 v/v) wasthen allowed to cool at room temperature and the solids ofN,N-diisopropylethylamine hydroiodide were filtered off. To theresulting filtrate was added ether (500 ml) and the solids were againfiltered off. The filtrate thus obtained was then evaporated to drynessto yield a red oil to which was then added 1N HCl (approx. 121 ml). Theresulting bilayer liquid was then extracted using 100 ml ether. Theether layer was allowed to stand at room temperature for 30 minutesresulting in a crystalline product.

The crude crystalline product was recrystallized in 165 mlmethanol:ether (1:10 v/v) to yield pure m-(N,N-diheptyl)aminophenolhydrochloride (6.0 g). Similarly, the crude product (2.0 g) from themother liquor was recrystallized from methanol:ether to yield 1.2 g ofpure m-(N,N-diheptyl)aminophenol hydrochloride. The total yield ofproduct was 7.2 g (69%, mp 110°-111°).

A. Synthesis

A 100 ml, three-neck flask was fitted with a Dean-Stark trap condenser,and maintained under positive argon pressure. The solid3-[N-ethyl-N-(3-hydroxyphenyl)amino]propane sulfonic acid, (906.5 mg,3.5 mmole) was added to the flask followed by 20 ml of an azeotropicallydried mixture of ethylene glycol and n-butanol (1:1). The mixture washeated in an oil bath until a clear solution was obtained.

The m-(N,N-diheptyl)aminophenol hydrochloride (1 g, 3.2 mole) wasdissolved in 10 ml of an azeotropically dried mixture of n-butanol andbenzene (1:2). Sodium bicarbonate (600 mg, 7 mmole) was slurried in themixture and transferred to the reaction vessel by Pasteur pipet.Vigorous bubbling from the evolution of carbon dioxide quickly subsided.The mixture was refluxed for 30 minutes to remove any traces of water.

Reflux was halted, and squaric acid (400 mg, 3.5 mmole), was added allat once to the still hot mixture followed by an additional 5 ml ofethylene glycol-n-butanol mixture. The reaction mixture was againbrought to reflux and blue color appeared within minutes. The reflux wasmaintained for approximately 7 hours at which time benzene was distilledoff and the reaction was allowed to cool. The reaction vessel wasstoppered and placed in the freezer for more than 48 hours.

The cold mixture was filtered to collect a quantity of dark,greenish-black crystals which were washed with cold n-butanol followedby methanol and then vacuum dried at 65° C. The recovered dye mixtureweighed 1.3 g.

B. Purification

The detergent dye mixture was transferred to an extraction thimble in aSoxhlet apparatus. Extraction with a mixture of methanol-methylenechloride removed the1,3-bis[4-(diheptylamino)-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediyliumdihydroxide, bis (inner salt), (THpS) and a small amount of C₇ SAS dye.Methylene chloride was removed by evaporation using a Rotovap, and theTHpS which crystallized from the residual methanol was removed byfiltration. The methanol filtrate was diluted with additional methanol(approximately 250 ml total volume) and used to continue the extraction.When the extraction was complete, the flask was removed from the Soxhletapparatus and the contents allowed to cool. Twenty-five grams (25 g) ofRP-8 silica gel were added with stirring followed by the dropwiseaddition of 150 ml of water. Stirring was stopped and the supernatantdecanted. The dark blue silica gel slurry was transferred to a columncontaining an additional 10 g RP-8 silica gel equilibrated with 40%water in methanol. The column was washed with 40% water in methanoluntil all1,3-bis[4-[N-ethyl-N-(3-sulfopropyl)-amino]-2-hydroxyphenyl]2,4-dihydroxycyclobutenediyliumdihydroxide, bis (inner salt) (BSAS) had been removed. The column wasthen washed with 30% water in methanol until C₇ SAS began to elute.Elution was completed with 20% water in methanol. The fractionscontaining the C₇ SAS dye were combined and stripped to dryness on theRotovap and the residue dissolved in methanol-methylene chloride (1:1).The methylene chloride was slowly distilled off and the methanol wasconcentrated. After cooling, the dark-blue mixture was placed in thefreezer at 0° C. overnight. Filtration and vacuum drying afforded 0.513g of analytically pure C₇ SAS as a greenish-black solid. The yield was44% of theoretical.

Elemental analysis gave C=61.73, H=7.54, N=4.06, Na 3.23, which isconsistent with the monohydrate of the sodium salt: SO₆ N₂ C₃₅ H₄₇ Na-H₂O.UV, λ_(max) =642 nm (MeOH).

EXAMPLE 3 Preparation of C₇ SAS Using Dimethyl sulfoxide (DMSO)

A. Synthesis

A mixture of m-(N,N-diheptyl)aminophenol hydrochloride (1) (770 mg, 2.25mmoles), 3-[N-ethyl-N-(3-hydroxyphenyl)amino]propane sulfonic acid (2)(678 mg, 2.5 mmoles), dihydroxy-3-cyclobutene-1,2-dione (3) (285 mg, 2.5mmoles, Aldrich Chemical Co., Inc.), DMSO (4 ml) and sodium bicarbonate(389 mg) in 40 ml of n-butanol-benzene (2:1 by volume, predried for 2hours using azeotrope) was refluxed under nitrogen with azeotropicremoval of water for two hours. The mixture was allowed to stand at roomtemperature under nitrogen overnight. To the product was added ether(500 ml), and the resulting precipitates were filtered and washed with50 ml of water. A crude preparation (370 mg) of the C₇ SAS detergent dye(4) was obtained.

B. Purification

The purification of the C₇ SAS detergent dye was as described in Example2. The properties of the C₇ SAS dye, i.e., the C₇ SAS dye shows minimalleaching and can stain cells in the presence of plasma, make it asuitable replacement for the DEAS dye used in the blood typing assays asdescribed in Examples 5, 6 and 7 as well as in antibody screening assaysand cross-match.

EXAMPLE 4 Preparation of1-[4-[N-ethyl-N-(3-sulfopropyl)amino]-2-hydroxyphenyl]-3-[4-dioctylamino-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediyliumdihydroxide, bis (inner salt) (C₈ SAS) ##STR5##

Preparation of starting materials:

m-(N,N-dioctyl)aminophenol hydrochloride, was prepared as follows:

A mixture of 3-aminophenol (5 g, 0.046 mole, Aldrich Chemical Co.,Inc.), 1-iodooctane (22 g, 0.092 moles, Aldrich Chemical Co., Inc.) andN,N-diisopropylethylamine (18 g, 0.139 mole, Aldrich Chemical Co., Inc.)in methanol (25 ml) was refluxed under nitrogen for 24 hours. An aliquotof the reaction product was spotted on TLC (silica, 20% EtOAc:hexane).The TLC showed formation of dialkylated and monoalkylated product,therefore, additional amounts of iodooctane (10 g, 0.042 mole) wereadded and the resulting mixture was refluxed for an additional 4 hours.

The reaction product (R_(f) 0.64, silica, 20% ethyl acetate:hexane wasconcentrated until a precipitate was formed. The resulting solids ofN,N-diisopropylethylamine hydroiodide were collected and washed withether. The filtration procedure was repeated one more time and theremaining filtrate was acidified using 1N HCl and extracted with ether.The ether layer was allowed to stand at room temperature until crystalsformed. The crystals were collected to yield 560 mg ofm-(N,N-dioctyl)aminophenol hydrochloride. The product was furtherpurified on preparative TLC (silica, 2:1 ethyl acetate/hexane) withrecovery of product by elution with 20% MeOH/CH₂ Cl₂ resulting in a pureproduct (400 mg, mp 112°-114°). For the purification of largerquantities of material, recrystallization from methanol-ether ispreferred.

The preparation of 3-[N-ethyl-N-(3-hydroxyphenyl)amino]propane sulfonicacid was as described in Example 2.

A. Synthesis

A mixture of m-(N,N-dioctyl)aminophenol hydrochloride (5) (600 mg, 1.62mmoles), 3-[N-ethyl-N-(3-hydroxyphenyl)amino]propane sulfonic acid (2)(648 mg, 2.5 mmoles), squaric acid (3) (285 mg, 2.5 mmoles, from AldrichChemical Co., Inc.), DMSO (4 ml), and sodium bicarbonate (410 mg) in 40ml of n-butanol-benzene (2:1 by volume, predried for 2 hours usingazeotrope) was refluxed using azeotrope under nitrogen for two hours. Itwas then allowed to stand at room temperature under nitrogen overnight.The resulting blue dye was filtered and the precipitate washed with 5 mlof 0.1N HCl followed by 1 ml of water to yield crude C₈ SAS squarainedye (6) (700 mg).

B. Purification

Method 1: The crude C₈ SAS squaraine dye (50 mg) was stirred in a smallamount of water (approximately 1 ml), and the resulting suspension wasfiltered and washed with water until there was complete removal of thedi-SO₃ squarate dye. The residual C₈ SAS dye was then heated in methanol(approximately 10 ml) and filtered. To the resulting hot filtrate wasthen added 10 ml of ether and the mixture was cooled to 5° C. Thismethod gave a yield of 7.8 mg of pure C₈ detergent dye. UV (DMSO,H⁺),λ_(max) =657.8 nm.

Method 2: The crude C₈ SAS detergent dye (120 mg) was dissolved inapproximately 5 ml of 20% MeOH/CH₂ Cl₂ and chromatographed on eightthick layer silica gel plates (Analtech Uniplate, silica gel GF, 20×20cm, 1000 microns, Catalog No. 02013). The band in the center (Rf 0.48)was scraped off and eluted, using 30% MeOH/CH₂ Cl₂ to yield 15 mg ofpurified C₈ -SAS detergent dye.

EXAMPLE 5 Assay for the Determination of the D (Rho) Blood Group Antigen

A saturated solution of DEAS in dimethylformamide (DMF) was prepared andthen diluted 1:10 (by volume) with DMF. Fifty μl of the diluted DEASsolution was mixed dropwise with 1 ml of a whole blood sample.Immediately thereafter, ten μl of this mixture was mixed with 10 μl ofantibody (commercially available typing reagent) specific for the D(Rh_(o)) blood group antigen. The mixture was held for one minute atambient temperature and then diluted with 1.5 ml of phosphate buffercontaining serum albumin and sucrose.

The medium was analyzed for a change in fluorescence as a result ofagglutination of cells by means of the limited volume method andapparatus for particle counting disclosed in U.S. Pat. No. 4,564,598issued Jan. 14, 1986.

The single fiber end of a "Y"-shaped fiber optics coupler obtained fromKaptron, Inc., Palo Alto, Calif. (Splitter-Monitor, Model FOMS-850-P),was submerged in the medium. The fiber had a diameter of 50 microns andproduced an excitation cone with a half angle of 12° and an effectivesampling volume of 1×10⁻⁷ ml. Excitation light from a He-Ne laser (632.9nm) was fed into one of the two branch fibers. The portion of thefluorescence emitted from the cells which entered the submerged fiberend was split at the fiber juncture to transmit equal halves back alongthe two branch fibers. The portion traveling through the second branchfiber was then read on a high-gain EMI photo-multiplier after filteringout interference within gate times of one millisecond at the rate of oneevery 0.1 second for periods of time ranging from 50 to 500 seconds. Theaverage number of fluorescent pulses per gate time was then determinedby computer. To obtain the mean fluorescence (x), variations in thefluorescent pulses per gate time were quantified by fluctuations or peakheight analysis.

Two types of control runs were made to establish a standard emissionlevel.

(a) Samples that were typed as D (Rh_(o)) negative by conventionaltyping were assayed in the same way.

(b) A commercially available "Rh control" reagent which includes all theingredients of a D (Rh₀) typing reagent except for the antibody was usedin the above assay in place of the antibody reagent.

The results from samples from five positive and five negativeindividuals are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Type             Signal*                                                      ______________________________________                                        D (Rh.sub.o) positive                                                                          84                                                                            108                                                                           72                                                                            46                                                                            74                                                           D (Rh.sub.o) neqative                                                                          18                                                                            23                                                                            22                                                                            17                                                                            20                                                           Control          21                                                                            19                                                                            17                                                           ______________________________________                                         *Signa1 was obtained by fluctuation ana1ysis as described in the              specification, Signals greater than 40 were regarded as positive.        

EXAMPLE 6 Assay for the Determination of the Antibody Specific for the ABlood Group Antigen

Whole type A blood was centrifuged at 2800 rpm and the supernatant andbuffy coat of the white cells were removed by aspiration. The packedcells were washed free of plasma using isotonic buffered saline andsuspended at 50% hematocrit in buffer containing 10% bovine serumalbumin.

A saturated solution of DEAS in DMF was prepared and diluted 1:10 (byvolume) with DMF. Fifty μl of the diluted DEAS solution was mixeddropwise under continuous vortexing with 1 ml of the above type A cellsuspension.

Ten μl of the above suspension was mixed with 20 μl of a whole bloodsample. The mixture was held for one minute at ambient temperaturediluted with 3 ml of buffer containing serum albumin and dextran, andanalyzed as described above in Example 5 using the limited volume methodand apparatus for particle counting. The results are summarized in Table2.

                  TABLE 2                                                         ______________________________________                                               Group*  Signal**                                                       ______________________________________                                               A       18                                                                            23                                                                            25                                                                            13                                                                            18                                                                    B       112                                                                           110                                                                           91                                                                            107                                                                           56                                                                    AB      35                                                                            25                                                                            19                                                                            22                                                                            18                                                                    O       106                                                                           134                                                                           81                                                                            116                                                                           80                                                                    Control***                                                                            15                                                                            18                                                                            16                                                             ______________________________________                                         *Samples from five separate individuals having the listed blood groups        were tested.                                                                  **Signal was obtained by fluctuation analysis as described in                 specification, Signals greater than 40 were regarded as positive.             ***Control signals were obtained by reacting the A cells with blood from      individuals who were known to be of AB type.                             

EXAMPLE 7

The assay of Example 5 was repeated for blood group antigens A, B, and Ousing antibody specific for the A(α A) and B(α B) blood group antigensand antibodies obtained from type O individuals (α A,B), respectively.The results are summarized in Table 3.

                  TABLE 3                                                         ______________________________________                                        Blood Type   Reagent       Signal*                                            ______________________________________                                        A            αA      103                                                             αB      21                                                              αA,B    74                                                              Control - no reagent                                                                        21                                                 B            αA      19                                                              αB      100                                                             αA,B    113                                                             Control - no reagent                                                                        22                                                 O            αA      18                                                              αB      22                                                              αA,B    17                                                              Control - no reagent                                                                        23                                                 ______________________________________                                         *Signal was obtained by fluctuation analysis as described in the              specification. Signals greater than 40 were regarded as positive.        

EXAMPLE 8 Staining of Reagent Red Blood Cells

A. C₇ SAS Detergent Dye in saline

Human erythrocytes were washed in isotonic saline and suspended toapproximately 4×10⁹ cells/ml in saline containing 20 g/l bovine serumalbumin (BSA).

Squaraine sulfonate dye, C₇ SAS, (100 ul, 10⁻⁴ M in dimethylacetamide)was added dropwise to 1 ml of saline containing BSA with continuousvortexing. The solution was immediately added to 1 ml of the cellsuspension, dropwise with continous vortexing. The resulting cellsuspension was then mixed gently. The cells were washed in isotonicsaline and stored at approximately 5×10⁸ cells/ml (5% hematocrit) forreverse grouping and 1×10⁹ cells/ml (10% hematocrit) for antibodyscreening, in an isotonic cell storage medium.

Fluorescence was determined by diluting 10 ul of 10% hematocrit cells in0.5 ml saline and measuring the mean fluorescence (x) as described inExample 5. Incorporation of fluorescence into cells over time isillustrated in Table 4.

                  TABLE 4                                                         ______________________________________                                               Time (min.)                                                                           - x                                                            ______________________________________                                                1       4                                                                    30      100                                                                   60      138                                                                   90      160                                                                   120     176                                                                   150     168                                                            ______________________________________                                    

An x value of approximately 100 was found to provide sufficientinstrument sensitivity and could be achieved within approximately 30minutes of mixing the cells with the dye in saline/BSA.

B. C₇ SAS in plasma 50 ul of C₇ SAS detergent dye (1×10⁻⁴ M indimethylacetamide) was mixed with 1 ml of whole blood. At the timeintervals shown in Table 5, a portion of the sample was centrifuged. Thecells were washed and resuspended in saline prior to fluorescencemeasurements. Table 5 shows the fluoresence (x) of the cells over time.

                  TABLE 5                                                         ______________________________________                                        Time (min.)  Fluorescence (- x)                                               ______________________________________                                         0            43                                                               11           55                                                               60          110                                                              180          176                                                              300          229                                                              ______________________________________                                    

C. C₈ SAS in saline

Red cells were washed in saline and suspended to 20% hematocrit insuspension medium (2% BSA, 2% pluronic or 0.4% β-cyclodextrin). To 1 mlof the cell suspension was added with vortexing 50 μl of 1×10⁻⁴ M C₈ SASdye solution in dimethylacetamide. The cells were allowed to stand atroom temperature for 10 minutes and were then washed in saline andsuspended in an isotonic cell storage medium. The resulting fluorescencein three different suspension media is shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Suspension media                                                                              Fluorescence (- x)                                            ______________________________________                                        2% BSA           41                                                           2% Pluronic     106                                                           4% β-cyclodextrin                                                                        109                                                           ______________________________________                                    

EXAMPLE 9 Loss of Dyes from Stained Cells During Assay Incubation

To measure the loss of fluorescence from stained cells during incubationwith sample at 37° C., the cells were stained with various squarainedyes and suspended to 0.9% by volume in a 1:1 (vol:vol) mixture ofplasma in a low ionic strength solution (LISS). The mixtures wereincubated in test tubes at 37° C. At selected time intervals, the testtubes were removed, the cells separated by centrifugation, washed insaline and resuspended in 0.5 ml saline. The fluorescence of thesuspensions (x) was measured as described in Example 8. Table 7 showsthe relative fluorescence of the cells when stained with the variousdyes.

                  TABLE 7                                                         ______________________________________                                                 Fluorescene (- x)                                                    Time (min) DEAS         C.sub.7 SAS                                                                           C.sub.8 SAS                                   ______________________________________                                         0         100          100     100                                            5         5            104     102                                           10         6            102     102                                           15         7             98      99                                           ______________________________________                                    

Table 7 shows the relatively slow rate of leaching of the detergentdyes, C₇ SAS and C₈ SAS compared to DEAS under actual antibody screenand cross-match assay conditions. DEAS stains cells very rapidly, asshown in Example 5, compared to the rate of staining of cells when C₇SAS is used (Example 8) and is therefore preferred in assays that do notrequire substantial dilution or separation of the stained cells.

EXAMPLE 10 Use of C₇ SAS Stained Cells in Reverse Grouping

10 μl (5% hematocrit) of cells stained with C₇ SAS dye was placed in areaction cup and 50 μl of the blood sample added. After mixing for 2minutes, the suspension was diluted with 0.5 ml saline and a measurementtaken to determine the agglutination index. The results are shown inTable 8.

                  TABLE 8                                                         ______________________________________                                        Agglutination Indices resulting from mixing cells                             stained with C.sub.7 SAS with blood samples having various                    AB0 Types:                                                                              Blood Type                                                                    B          A       AB                                               ______________________________________                                        Type B Cells                                                                              21            921    60                                                       66           11752   15                                                       69            9138   98                                                       37           13336   83                                                       64           10750   34                                                       59            4066                                                Type A Cells                                                                              19494         121    ND                                                       17266          85    ND                                                       14666          72    ND                                                       19921                ND                                           ______________________________________                                    

Table 8 shows that standard B cells are agglutinated with blood thatdoes not have the B antigen and therefore, has anti-B-antibodies.Stained A cells are agglutinated with blood that does not have the Aantigen and therefore, has anti-A-antibodies. The use of the DEAS dye isnot effective in this particular type of assay due to the leaching outof the dye and resulting loss of signal.

EXAMPLE 11 Use of C₇ SAS Stained Cells for Antibody Screen Assays

For each sample, 10 μl of 10% hematocrit screen cells stained with C₇SAS dye by the methods previously described was incubated with 50 μl ofeither fresh or previously frozen plasma and 50 μl of LISS, for 10minutes at 37° C. in a glass test tube. The samples were washed withLISS by centrifugation or magnetic separation. The resulting cellsuspension was diluted to 40 μl with LISS and applied to a latexreaction block with 10 μl of anti-human globulin-PVP reagent. Anautomated protocol was used which mixed the reactants then diluted thesample and measured the agglutination index. Table 9 showsrepresentative agglutination indices (AI) values for positive andnegative samples.

                  TABLE 9                                                         ______________________________________                                        Antibody                 AI                                                   ______________________________________                                        Anti-E         positive  15200                                                               negative  220                                                  Anti-Fy.sup.a  positive  1302                                                                negative  115                                                  Anti-Jk.sup.a  positive  804                                                                 negative  166                                                  ______________________________________                                    

The above data demonstrate that the method of the invention has utilityfor assaying for a wide variety of analytes and has particular utilityin blood typing. The method is simple and rapid.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes or modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A compound of the formula: ##STR6## wherein: R₁ and R₂ are independently selected from the group consisting of lower alkyl (1-5 carbon atoms) with the proviso that at least one of the R₁ and R₂ groups has a substituent selected from the group consisting of --SO₃ H, --OSO₃ H, --PO₃ H₂, --OPO₃ H₂, --COOH and --NHSO₃ H;R₃ and R₄ are independently selected from the group consisting of alkyl (5-15 carbon atoms); and R₅ and R₆ are independently selected from the group consisting of hydrogen, hydroxyl, and lower alkoxyl (1-5 carbon atoms).
 2. The compound of claim 1 wherein the substituent of R₁ and R₂ is --SO₃ H.
 3. The compound of claim 1 wherein R₁ is lower alkyl (1-5 carbon atoms); R₃ and R₄ are alkyl (5-15 carbon atoms); and R₂ is --(CH₂)_(n) SO₃ H, where n is 2-5.
 4. The compound of claim 3 wherein R₁ is ethyl, R₃ and R₄ are heptyl and n is
 3. 